Orthologous proteins of species adapted to different temperatures exhibit differences in stability and function that are interpreted to reflect adaptive variation in structural “flexibility.” ...However, quantifying flexibility and comparing flexibility across proteins has remained a challenge. To address this issue, we examined temperature effects on cytosolic malate dehydrogenase (cMDH) orthologs from differently thermally adapted congeners of five genera of marine molluscs whose field body temperatures span a range of ∼60 °C. We describe consistent patterns of convergent evolution in adaptation of function temperature effects on K
M of cofactor (NADH) and structural stability (rate of heat denaturation of activity). To determine how these differences depend on flexibilities of overall structure and of regions known to be important in binding and catalysis, we performed molecular dynamics simulation (MDS) analyses. MDS analyses revealed a significant negative correlation between adaptation temperature and heat-induced increase of backbone atom movements root mean square deviation (rmsd) of main-chain atoms. Root mean square fluctuations (RMSFs) of movement by individual amino acid residues varied across the sequence in a qualitatively similar pattern among orthologs. Regions of sequence involved in ligand binding and catalysis—termed mobile regions 1 and 2 (MR1 and MR2), respectively—showed the largest values for RMSF. Heat-induced changes in RMSF values across the sequence and, importantly, in MR1 and MR2 were greatest in cold-adapted species. MDS methods are shown to provide powerful tools for examining adaptation of enzymes by providing a quantitative index of protein flexibility and identifying sequence regions where adaptive change in flexibility occurs.
ABSTRACT
Elucidating the physiological mechanisms that underlie thermal stress and discovering how species differ in capacities for phenotypic acclimatization and evolutionary adaptation to this ...stress is critical for understanding current latitudinal and vertical distribution patterns of species and for predicting their future state in a warming world. Such mechanistic analyses require careful choice of study systems (species and temperature‐sensitive traits) and design of laboratory experiments that reflect the complexities of in situ conditions. Here, we critically review a wide range of studies of intertidal molluscs that provide mechanistic accounts of thermal effects across all levels of biological organization – behavioural, organismal, organ level, cellular, molecular, and genomic – and show how temperature‐sensitive traits govern distribution patterns and capacities for coping with thermal stress. Comparisons of congeners from different thermal habitats are especially effective means for identifying adaptive variation. We employ these mechanistic analyses to illustrate how species differ in the severity of threats posed by rising temperature. Counterintuitively, we show that some of the most heat‐tolerant species may be most threatened by increases in temperatures because of their small thermal safety margins and minimal abilities to acclimatize to higher temperatures. We discuss recent molecular biological and genomic studies that provide critical foundations for understanding the types of evolutionary changes in protein structure, RNA secondary structure, genome content, and gene expression capacities that underlie adaptation to temperature. Duplication of stress‐related genes, as found in heat‐tolerant molluscs, may provide enhanced capacity for coping with higher temperatures. We propose that the anatomical, behavioural, physiological, and genomic diversity found among intertidal molluscs, which commonly are of critical importance and high abundance in these ecosystems, makes this group of animals a highly appropriate study system for addressing questions about the mechanistic determinants of current and future distribution patterns of intertidal organisms.
A quantitative understanding of physiological thermal responses is vital for forecasting species distributional shifts in response to climate change. Many studies have focused on metabolic rate as a ...global metric for analyzing the sublethal effects of changing environments on physiology. Thermal performance curves (TPCs) have been suggested as a viable analytical framework, but standard TPCs may not fully capture physiological responses, due in part to failure to consider the process of metabolic depression. We derived a model based on the nonlinear regression of biological temperature‐dependent rate processes and built a heart rate data set for 26 species of intertidal molluscs distributed from 33°S to ~40°N. We then calculated physiological thermal performance limits with continuous heating using T1/2H, the temperature at which heart rate is decreased to 50% of the maximal rate, as a more realistic measure of upper thermal limits. Results indicate that heat‐induced metabolic depression of cardiac performance is a common adaptive response that allows tolerance of harsh environments. Furthermore, our model accounted for the high inter‐individual variability in the shape of cardiac TPCs. We then used these TPCs to calculate physiological thermal safety margins (pTSM), the difference between the maximal operative temperature (95th percentile of field temperatures) and T1/2H of each individual. Using pTSMs, we developed a physiological species distribution model (pSDM) to forecast future geographic distributions. pSDM results indicate that climate‐induced species range shifts are potentially less severe than predicted by a simple correlative SDM. Species with metabolic depression below the optimum temperature will be more thermal resistant at their warm trailing edges. High intraspecific variability further suggests that models based on species‐level vulnerability to environmental change may be problematic. This multi‐scale, mechanistic understanding that incorporates metabolic depression and inter‐individual variability in thermal response enables better predictions about the relationship between thermal stress and species distributions.
This multi‐scale, mechanistic understanding that incorporates metabolic depression and inter‐individual variability in thermal response enables better predictions about the relationship between thermal stress and species distributions.
Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and ...function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from −1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme’s thermal responses and foster evolutionary adaptation of function.
Macromolecular function commonly involves rapidly reversible alterations in three-dimensional structure (conformation). To allow these essential conformational changes, macromolecules must possess ...higher order structures that are appropriately balanced between rigidity and flexibility. Because of the low stabilization free energies (marginal stabilities) of macromolecule conformations, temperature changes have strong effects on conformation and, thereby, on function. As is well known for proteins, during evolution, temperature-adaptive changes in sequence foster retention of optimal marginal stability at a species' normal physiological temperatures. Here, we extend this type of analysis to messenger RNAs (mRNAs), a class of macromolecules for which the stability-lability balance has not been elucidated. We employ in silico methods to determine secondary structures and estimate changes in free energy of folding (ΔG
) for 25 orthologous mRNAs that encode the enzyme cytosolic malate dehydrogenase in marine mollusks with adaptation temperatures spanning an almost 60 °C range. The change in free energy that occurs during formation of the ensemble of mRNA secondary structures is significantly correlated with adaptation temperature: ΔG
values are all negative and their absolute values increase with adaptation temperature. A principal mechanism underlying these adaptations is a significant increase in synonymous guanine + cytosine substitutions with increasing temperature. These findings open up an avenue of exploration in molecular evolution and raise interesting questions about the interaction between temperature-adaptive changes in mRNA sequence and in the proteins they encode.
Background
Dysregulation of eicosanoids is associated with asthma and a composite of oxylipins, including exhaled leukotriene B4 (LTB4), characterizes childhood asthma. While fractional exhaled ...nitric oxide (FeNO) has been used as the standard for monitoring steroid responsiveness, the potential utility of eicosanoids in monitoring the therapeutic outcomes remains unclear. We aimed to examine the levels of major eicosanoids representing different metabolic pathways in exhaled breath condensates (EBCs) of children with asthma during exacerbation and after treatment.
Methods
Levels of 6 exhaled eicosanoid species in asthmatic children and healthy subjects were evaluated using ELISA.
Results
In addition to those previously reported, including LTB4, the levels of exhaled 15‐hydroxyeicosatetraenoic acid (15‐HETE), but not thromboxane B2 (TXB2), showed significant difference between asthmatics (N = 318) and healthy controls (N = 97), particularly the severe group showed the lowest levels of exhaled 15‐HETE. Receiver operating characteristic (ROC) curve analyses revealed similar distinguishing power for the levels of 15‐HETE, FEV1 (forced expiratory volume in the first second), and FeNO, while the 15‐HETE/LTB4 ratio was significantly lower in subjects with asthma as compared to that of healthy controls (p < 0.0001). Analysis of asthmatics (N = 75) during exacerbation and convalescence showed significant improvement in lung function (FEV1, p < .001), but not FeNO, concomitant with significantly increased levels of 15‐HETE (p < .001) and reduced levels of TXB2 (p < .05) at convalescence, particularly for those who at the top 30% level during exacerbation. Further, decreased LTB4 and lipoxin A4 (LXA4) at convalescence were noted only in those at the top 30 percentile during exacerbation.
Conclusion
The exhaled 15‐HETE was found to discriminate childhood asthma while decreased levels of exhaled TXB2 and increased levels of 15‐HETE were prominent at convalescence.
Background
This study aimed to investigate whether maternal allergy is associated with soluble CD14 (sCD14) and fatty acid composition in different stages of lactation and the onset of atopic ...dermatitis (AD) in early childhood.
Methods
In total, 443 mother‐child groups (445 children) were enrolled in the Prediction of Allergies in Taiwanese Children birth cohort study. Colostrum and mature milk at 2 months postpartum (2‐month HM) were collected from lactating mothers. Information regarding parental allergy histories and physician‐diagnosed atopic diseases was obtained using age‐specific questionnaires (0‐2 years). We compared sCD14 levels and the composition of 30 fatty acids in the colostrum and 2‐month HM, respectively, between allergic and non‐allergic mothers and between children with and without AD by the age of 2 years.
Results
In total, 185 (41.8%) mothers presented with allergies, and 154 (34.6%) children had physician‐diagnosed AD by the age of 2 years. Both in the colostrum and 2‐month HM of 289 lactating mothers, sCD14 levels were significantly lower in allergic mothers whose children presented with AD compared with children who did not (P = 0.015 and 0.044, respectively). Among the children with AD who were born to non‐allergic mothers, sCD14 levels were lower. However, the result was not statistically significant (P = 0.376 and 0.264, respectively). Our data revealed the lack of associations between fatty acid composition and AD (P > 0.05).
Conclusion
Decreased sCD14 levels in the colostrum and 2‐month HM were associated with AD at 2 years of age, particularly among children born to mothers with allergies.
Gallic acid (GA) is a simple polyphenol found in food and traditional Chinese medicine. Here, we determined the effects of GA administration in a combined mouse model of high-fat diet (HFD)-induced ...obesity and low-dose streptozotocin (STZ)-induced hyperglycemia, which mimics the concurrent non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes pathological condition. By combining the results of physiological assessments, pathological examinations, metabolomic studies of blood, urine, liver, and muscle, and measurements of gene expression, we attempted to elucidate the efficacy of GA and the underlying mechanism of action of GA in hyperglycemic and dyslipidemic mice. HFD and STZ induced severe diabetes, NAFLD, and other metabolic disorders in mice. However, the results of liver histopathology and serum biochemical examinations indicated that daily GA treatment alleviated the high blood glucose levels in the mice and decelerated the progression of NAFLD. In addition, our results show that the hepatoprotective effect of GA in diabetic mice occurs in part through a partially preventing disordered metabolic pathway related to glucose, lipids, amino acids, purines, and pyrimidines. Specifically, the mechanism responsible for alleviation of lipid accumulation is related to the upregulation of
-oxidation and ketogenesis. These findings indicate that GA alleviates metabolic diseases through novel mechanisms.
Overexpression of SH2-containing-5'-inositol phosphatase-2 (SHIP2) correlates with poor survival in breast cancer. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here, we ...showed that the percentage of SHIP2(+) cells was positively correlated with that of CD24(-) CD44(+) cells in 60 breast cancer specimens. Among 20 estrogen receptor (ER)-negative samples, 17 had greater SHIP2 expression in CD24(-) CD44(+) subpopulation than the remaining subpopulation. Data mining of microarray analysis of 295 breast tumors showed a significant correlation of higher SHIP2 expression with distant metastasis. Examination of patient-derived mouse xenografts revealed that SHIP2 protein and its tyrosine 1135 phosphorylation were significantly higher in BCSCs, identified as CD24(-) CD44(+) or aldehyde dehydrogenase (ALDH(+)), than non-BCSCs. SHIP2 silencing or inhibitor of SHIP2 phosphatase significantly decreased mammosphere-forming efficiency, ALDH(+) subpopulation in vitro and tumorigenicity of BCSCs in vivo. Overexpression of SHIP2 enhanced the expression of epithelial-mesenchymal transition markers including vimentin (VIM), which was mainly expressed in ER-negative breast cancer cells with higher level in mammospheres than monolayer culture. Ablation of c-Jun N-terminal kinase 1 (JNK1), JNK2, or VIM diminished the increased ALDH(+) population and tumorigenicity, induced by SHIP2 overexpression. BCSCs displayed greater expression of phospho-JNK than non-BCSCs and silencing of JNK suppressed SHIP2-mediated upregulation of VIM. Furthermore, SHIP2 overexpression enhanced Akt activation, but Akt inhibition failed to influence SHIP2-induced phospho-JNK/VIM upregulation. In conclusion, SHIP2 plays a key role in BCSCs of ER-negative breast cancers through activation of Akt and JNK with upregulation of VIM and may serve as a target for therapy directed at BCSCs.
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